Use of M4 Metalloprotease in Wort Production

ABSTRACT

A method of preparing a wort with an increased level of free amino nitrogen (FAN) comprising: a) preparing a mash from a grist comprising malt and/or adjunct; and b) adding a M4 mettalloprotease obtainable from Actinobacteria; and wherein the amount of free amino nitrogen (FAN) in the wort is increased as compared to a wort produced in the absence of the M4 metalloprotease.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to wort making for brewing and non-alcoholic beverages. More particularly, it relates to a method of increasing the level of free amino nitrogen in a wort.

BACKGROUND OF THE INVENTION

Modern breweries need to have a high level of raw material flexibility with respect to adjunct inclusion and malt quality.

When adjuncts like corn grits, barley or rice are used in the brewing process instead of malt, or when under-modified low quality malt is used, this will result in a level of free amino nitrogen that is insufficient to have proper yeast fermentation.

Wort nitrogen is normally determined as FAN (free amino nitrogen). FAN includes all free primary amines and thus also includes amines of nucleotides and other compounds which are not amino acids.

The inventors have found a surprisingly good method of increasing the level of FAN in a wort.

SUMMARY OF THE INVENTION

Surprisingly, it has been found that it is possible to significantly increase the level of FAN in a wort, so we claim:

-   A method of preparing a wort with an increased level of free amino     nitrogen (FAN) comprising:

a) preparing a mash from a grist comprising malt and/or adjunct; and

b) adding an M4 metalloprotease obtainable from Actinobacteria; and

wherein the amount of free amino nitrogen (FAN) in the wort is increased as compared to a wort produced in the absence of the M4 metalloprotease.

In one embodiment, the M4 metalloprotease has at least 80% sequence identity to the mature polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

In one embodiment, the M4 metalloprotease is a variant of the polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, comprising a substitution, deletion, and/or insertion at one or more positions.

In one embodiment, the M4 metalloprotease consists of the polypeptide of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

In one embodiment, the M4 metalloprotease according to the invention is added to the mash or to the wort.

In one embodiment, additionally an alpha amylase is added to the mash.

In one embodiment, additionally a beta glucanase is added to the mash.

In one embodiment, additionally a pullulanase is added to the mash.

In one embodiment, additionally a xylanase is added to the mash.

In one embodiment, additionally a lipase is added to the mash.

In one embodiment, the M4 metalloprotease is added in an amount of 1-100 mg enzyme protein per kg grist.

In one embodiment, the grist comprises at least 10% (w/w) adjunct.

In one embodiment, the adjunct is selected from the group consisting of barley, rice, corn, wheat, sorghum and cassava.

In one embodiment, the wort is fermented to obtain a beer.

In one embodiment, the mashing is done in the absence of a protein rest.

In one embodiment, the amount of free amino nitrogen (FAN) is increased by at least 20% when the M4 metalloprotease according to the invention is added in an amount of 10 mg Enzyme Protein per kg grist as compared to a wort produced in the absence of the M4 metalloprotease according to the invention.

The invention also describes use of an M4 metalloprotease obtainable from Actonobacteria in wort production.

DEFINITIONS Polypeptides Having Protease Activity

Polypeptides having protease activity, or proteases, are sometimes also designated peptidases, proteinases, peptide hydrolases, or proteolytic enzymes.

The term “protease” is defined herein as an enzyme that hydrolyses peptide bonds. This definition of protease also applies to the protease-part of the terms “parent protease” and “protease variant,” as used herein. The term “protease” includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, Calif., including supplements 1-5 published in Eur. J. Bio-chem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650. The nomenclature is regularly supplemented and updated; see, e.g., the World Wide Web (WWW) at http://www.chem.qmw.ac.uk/iubmb/enzyme/index.html.

Protease activity can be measured using any assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question. Assay-pH and assay-temperature are likewise to be adapted to the protease in question. Examples of assay-pH-values are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay-temperatures are 15, 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95° C. Examples of protease substrates are casein, such as Azurine-Crosslinked Casein (AZCL-casein).

Protease activity: The term “protease activity” means a proteolytic activity that catalyzes the hydrolysis of amide bond or a protein by hydrolysis of the peptide bond that link amino acids together in a polypeptide chain. Several assays for determining protease activity are available in the art. For purposes of the present invention, protease activity may be determined using Protazyme AK tablet (cross-linked and dyed casein; from Megazyme).

Allelic variant: The term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Catalytic domain: The term “catalytic domain” means the region of an enzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

Fragment: The term “fragment” means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has protease activity.

Host cell: The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having protease activity.

Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.

Operably linked: The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide in such a way that the control sequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment).

For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment).

Subsequence: The term “subsequence” means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5′ and/or 3′ end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having protease activity.

Variant: The term “variant” means a polypeptide having protease activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions, e.g., a substitution, insertion, and/or deletion at 10 positions; or a substitution, insertion, and/or deletion at 9 positions; or a substitution, insertion, and/or deletion at 8 positions; or a substitution, insertion, and/or deletion at 7 positions; or a substitution, insertion, and/or deletion at 6 positions; or a substitution, insertion, and/or deletion at 5 positions; or a substitution, insertion, and/or deletion at 4 positions; or a substitution, insertion, and/or deletion at 3 positions; or a substitution, insertion, and/or deletion at 2 positions; or a substitution, insertion, and/or deletion at 1 position.

A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding one or more (e.g. several) amino acids, e.g., 1-5 amino acids adjacent to and immediately following the amino acid occupying a position.

Beverage: The term beverage as used herein has the conventional meaning in the art and includes, but is not limited to beer, and any wort based beverage.

Beer: The term “beer” as used herein is intended to cover at least beer prepared from mashes prepared from unmalted cereals as well as all mashes prepared from malted cereals, and all mashes prepared from a mixture of malted and unmalted cereals. The term “beer” also covers beers prepared with adjuncts, and beers with all possible alcohol contents.

Grist: The term “grist” is understood as the starch or sugar containing material that is the basis for beer production, e.g., the barley malt and/or the adjunct. Generally, the grist does not contain any added water.

Malt: The term “malt” is understood as any malted cereal grain, in particular barley.

Adjunct: The term “adjunct” is understood as the part of the grist which is not barley malt. The adjunct may be any starch rich plant material, e.g., unmalted grain, such as, but not limited to, barley, corn, rice, sorghum, wheat and cassava, and also includes readily fermentable sugar and/or syrup. The starch of some of the adjuncts has a relatively low gelatinization temperature which enable them to be mashed in together with the malt, whereas other adjuncts such as rice, corn and sorghum has a higher gelatinization temperature, such adjuncts are typically separately cooked and liquefied with an alpha-amylase before they are added to the mash.

Mash: The term “mash” is understood as a starch containing slurry comprising crushed barley malt, and/or crushed unmalted grain, and/or other starch containing material, or a combination hereof, steeped in water to make wort.

Protein rest: The term “protein rest” is understood as a part of the mashing process.

Typically, it is the first step in mashing. It is performed at a temperature of around 45° C. to 55° C. for normally 15 to 30 minutes.

Wort: The term “wort” is understood as the unfermented liquor run-off following extracting the grist during mashing.

DETAILED DESCRIPTION OF THE INVENTION

The advantage of the present invention is that it allows the breweries to have a higher level of raw material flexibility with respect to adjunct inclusion and malt quality.

When adjuncts like corn grits, barley, or rice are included in the brewing process instead of malt, the level of FAN (free amino nitrogen) will be insufficient to have proper yeast fermentation. The same issue occurs when an under-modified low quality malt is used.

During traditional mashing, the endogenous malt proteases are capable of increasing the overall FAN level. This increase occurs mainly during the protein rest (e.g., 20 min, 50° C.).

Adding the M4 metalloprotease according to the invention to the mash may allow the breweries to eliminate the protein rest without losing FAN. Eliminating the protein rest will save time and energy in the brewing process and also minimize the lipoxygenase (LOX) catalyzed lipid oxidation leading to off-flavors in the final product.

Wort Production

The present invention relates to a method of producing a wort with an increased level of FAN, wherein an M4 metalloprotease obtainable from Actinobacteria, in particular an M4 metalloprotease that has at least 80% sequence identity to the polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, has been added to the mash or the wort.

In a preferred embodiment, the present invention relates to a method of producing a wort with an increased level of FAN, wherein an M4 metalloprotease that has at least 80% sequence identity to the mature polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, has been added to the mash or the wort.

The mash is obtainable by grinding a grist comprising malt and/or adjunct. Water may preferably be added to the grist, and is normally preheated in order for the mash to attain the desired mash temperature at the moment of mash forming. If the temperature of the formed mash is below the desired mashing temperature, additional heat is preferably supplied in order to attain the desired process temperature.

The temperature profile of the mashing process may be a profile from a conventional mashing process wherein the temperatures are set to achieve optimal degradation of the grist dry matter by the malt enzymes and/or the added enzymes.

The malt is preferably derived from one or more of the grains selected from the list consisting of, e.g., corn, barley, wheat, rye, sorghum, millet and rice, or any mixtures thereof.

Preferably, the malt is barley malt. The grist preferably comprises from 0.5% to 99% (w/w) malt, e.g., from 1% to 95% (w/w) malt, e.g., from 5% to 90% (w/w) malt, e.g., from 10% to 85% (w/w) malt, e.g., from 15% to 80% (w/w) malt, e.g., from 20% to 75% (w/w) malt, from 25% to 70% (w/w) malt, e.g., from 30% to 65% (w/w) malt.

In addition to malted grain, the grist may comprise one or more adjuncts such as unmalted corn, or other unmalted grain, such as barley, wheat, rye, oat, corn, rice, milo, millet and/or sorghum, or raw and/or refined starch and/or sugar containing material derived from plants like wheat, rye, oat, corn, rice, milo, millet, sorghum, pea, potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and/or sugar cane. According to the present invention, adjuncts may be obtained from tubers, roots, stems, leaves, legumes, cereals and/or whole grain.

Preferred is adjunct obtained from barley, corn, rice, wheat, sorghum and/or cassava; e.g., rice starch, corn starch, and/or corn grits.

The grist comprises typically from 1% to 80% (w/w) adjunct, e.g., from 5% to 75% (w/w) adjunct, e.g., from 10% to 70% (w/w) adjunct; in a particular embodiment the grist comprises at least 10% (w/w) adjunct. In a preferred embodiment, the grist comprises from 30% to 70% (w/w) adjunct.

In one aspect, the M4 metalloprotease according to the invention is introduced at the beginning of mashing. In another aspect, the M4 metalloprotease according to the invention is introduced during mashing. In another aspect, the M4 metalloprotease according to the invention is added to the wort.

The amount of added M4 metalloprotease according to the invention generally depends on various factors. For purposes of this invention, the amount of M4 metalloprotease used will generally be of from 0.1 mg to 100 mg EP (Enzyme Protein) per kg grist, preferably from 1 mg to 100 mg EP (Enzyme Protein) per kg grist; preferably from 1 mg to 50 mg EP (Enzyme Protein) per kg grist.

In a preferred embodiment, the amount of free amino nitrogen in the wort is increased by at least 20% (when the protease according to the invention is added in an amount of 10 mg Enzyme Protein per kg grist) as compared to a wort produced in the absence of the protease according to the invention, e.g., the amount of free amino nitrogen in the wort is increased by at least 30% (when the protease according to the invention is added in an amount of 10 mg Enzyme Protein per kg grist) as compared to a wort produced in the absence of the protease according to the invention, e.g., the amount of free amino nitrogen in the wort is increased by at least 40% (when the protease according to the invention is added in an amount of 10 mg Enzyme Protein per kg grist) as compared to a wort produced in the absence of the protease according to the invention, e.g., the amount of free amino nitrogen in the wort is increased by at least 50% (when the protease according to the invention is added in an amount of 10 mg Enzyme Protein per kg grist) as compared to a wort produced in the absence of the protease according to the invention.

In another preferred embodiment, a further enzyme(s) is added to the mash, said enzyme(s) including but not limited to alpha amylase, isoamylase, maltogenic amylase, protease, cellulase, beta glucanase, pullulanase, laccase, xylanase, lipase, phospholipase, phytase, and esterase.

In one aspect of the method, the further enzyme added includes a pullulanase.

In one aspect of the method, the further enzyme added includes an amylase, preferably an alpha amylase.

In one aspect of the method, the further enzyme added includes a beta glucanase.

In one aspect of the method, the further enzyme added includes a xylanase.

In one aspect of the method, the further enzyme added includes a lipase.

Following the separation of the wort from the spent grains of the grist, the wort may be used as it is or it may be dewatered to provide a concentrated and/or dried wort. The concentrated and/or dried wort may be used as brewing extract, as malt extract flavoring, for non-alcoholic malt beverages, malt vinegar, breakfast cereals, for confectionary, etc.

In a preferred embodiment, the wort is fermented to produce an alcoholic beverage, preferably a beer, e.g., ale, strong ale, bitter, stout, porter, lager, export beer, malt liquor, barley wine, happoshu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.

Fermentation of the wort may include pitching the wort with a yeast slurry comprising fresh yeast, i.e., yeast not previously used or the yeast may be recycled yeast. The yeast applied may be any yeast suitable for beer brewing, especially yeasts selected from Saccharomyces spp. such as S. cerevisiae and S. uvarum, including natural or artificially produced variants of these organisms.

The methods for fermentation of wort for production of beer are well known to the person skilled in the art.

Actinobacteria

Actinobacteria are a specific class of bacteria. Morphologically, they often resemble fungi because of their elongated cells that branch into filaments or hyphae.

Actinobacteria comprise the orders of Propionibacteriales, Streptomycetales, and Micromonosporales, which are further subdivided into families and genera.

Actinobacteria comprise the genera Streptomyces, Nocardia, Kitasatospora, Actinoplanes, Dactylosporangium, Micromonospora, Kribbella, Nocardioides, Propionibacterium, Nocardiopsis, Streptomonospora, and Thermobifida.

The M4 metalloprotease according to the present invention is obtainable from Actinobactera; in particular the M4 metalloprotease is selected from the group consisting of Streptomyces, Nocardia, Kitasatospora, Actinoplanes, Dactylosporangium, Micromonospora, Kribbella, Nocardioides, Propionibacterium, Nocardiopsis, Streptomonospora, and Thermobifida.

Proteases According to the Invention

The M4 metalloprotease according to the present invention is obtainable from Actinobacteria; in particular the M4 metalloprotease is obtainable from Streptomyces, Nocardia, Kitasatospora, Actinoplanes, Dactylosporangium, Micromonospora, Kribbella, Nocardioides, Propionibacterium, Nocardiopsis, Streptomonospora, or Thermobifida.

A protease may be classified as an M4 meatalloprotease as known in the art, e.g., by using the MEROPS database:

-   http://merops.sanger.ac.uk/cgi-bin/blast/submitblast/merops/advanced

The MEROPS database is described in “Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors”; Rawlings N D, Barrett A J, Finn R.; Nucleic Acids Res; 2016 Jan. 4; 44(D1):D343-50.

In one embodiment, the present invention relates to an isolated polypeptide having a sequence identity to the polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11 of at least 80%, e.g., of at least 81%, e.g., of at least 82%, e.g., of at least 83%, e.g., of at least 84%, e.g., of at least 85%, e.g., of at least 86%, e.g., of at least 87%, e.g., of at least 88%, e.g., of at least 89%, e.g., of at least 90%, e.g., of at least 91%, e.g., of at least 92%, e.g., of at least 93%, e.g., of at least 94%, e.g., of at least 95%, e.g., of at least 96%, e.g., of at least 97%, e.g., of at least 98%, e.g., of at least 99%, or 100% identical, and wherein the polypeptide has protease activity.

In one embodiment, the present invention relates to an isolated polypeptide having a sequence identity to the mature polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,

SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11 of at least 80%, e.g., of at least 81%, e.g., of at least 82%, e.g., of at least 83%, e.g., of at least 84%, e.g., of at least 85%, e.g., of at least 86%, e.g., of at least 87%, e.g., of at least 88%, e.g., of at least 89%, e.g., of at least 90%, e.g., of at least 91%, e.g., of at least 92%, e.g., of at least 93%, e.g., of at least 94%, e.g., of at least 95%, e.g., of at least 96%, e.g., of at least 97%, e.g., of at least 98%, e.g., of at least 99%, or 100% identical, and wherein the polypeptide has protease activity.

A polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; or is an allelic variant thereof; or is a fragment thereof having protease activity.

In another embodiment, the present invention relates to an isolated polypeptide having protease activity encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, or high stringency conditions with the polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, or the full-length complement thereof (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

In another embodiment, the present invention relates to variants of the polypeptide of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.

In one embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the polypeptide of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, is not more than 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope, or a binding domain.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708.

The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photo-affinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides.

Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Enzyme Compositions

The present invention also relates to compositions comprising the M4 metalloprotease of the present invention for use in wort production.

The compositions according to the invention may comprise the M4 metalloprotease of the present invention as the major enzymatic component, e.g., a mono-component composition.

Alternatively, the compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, amylase, alpha amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, beta glucanase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, peroxidase, phytase, polyphenoloxidase, pullulanase, ribonuclease, transglutaminase, or xylanase.

Preferably, the composition for use in wort production may comprise an M4 metalloprotease obtainable from Actinobacteria; in particular an M4 metalloprotease having at least 80% sequence identity to the polypeptide of SEQ ID NO: 1; SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; or the composition for use in wort production may comprise the M4 metalloprotease according to the invention and one or more enzymes selected from the group consisting of alpha amylase, beta glucanase, pullulanase, xylanase, and lipase.

The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. The compositions may be stabilized in accordance with methods known in the art. The dosage of the composition and other conditions under which the composition is used may be determined on the basis of methods known in the art.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLE 1 Cloning and Expression of M4 Metalloproteases from Bacterial Strains from the Class Actinobacteria

The metalloproteases were derived from bacterial strains obtained by isolation from environmental sources collected from the locations listed in Table 1.

The strain Streptomyces thermoalcalitolerans was obtained from the German collection of Microorganisms and Cell Cultures (DSMZ) as DSM41741 type strain of Streptomyces thermoalcalitolerans. This strain was originally isolated from garden soil in Yogyakarta, Indonesia (Kim et al., Classification of thermophilic streptomycetes, including the description of Streptomyces thermoalcalitolerans sp. nov., Int. J. Syst. Bacteriol. (1999) 49: 7-17).

TABLE 1 Organism Country of isolation Micromonospora sp.-61168 United Kingdom Streptomyces thermoalcalitolerans Indonesia Streptomyces ginsengisoli Japan Kribbella aluminosa China Streptomyces sp.-61982 Japan Streptomyces champavatii United Kingdom Streptomyces kathirae United Kingdom Streptomyces sp.-62237 Japan

Chromosomal DNA from pure cultures of the individual strains were purified and subjected to full genome sequencing using Illumina sequencing technology. The assembled genome sequences and subsequent analysis of the 16S ribosomal subunit gene sequences against the ARB-SILVA database (The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Quast et al. Nucl. Acids Res. 41 (2013): D590-D596) confirmed the identity of the strains.

The individual genome sequences were analyzed for metalloproteases from the MEROPS family M4 by comparison to the M4 protease NprE from B. subtilis 168 (Uniprot P68736) by search using the BLAST program.

This analysis identified genes encoding M4 metalloproteases with the nucleotide sequences given in SEQ ID NO: 12 to 22 and translated proteins sequences in SEQ ID NO:1 to SEQ ID NO:11.

The genes encoding the M4 metalloproteases was amplified from genomic DNA by PCR and fused with regulatory elements and homology regions for recombination into the B. subtilis genome.

The linear integration construct was a SOE-PCR fusion product (Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. and Pease, L. R. (1989) Engineering hybrid genes without the use of restriction enzymes, gene splicing by overlap extension Gene 77: 61-68) made by fusion of the gene between two Bacillus subtilis chromosomal regions along with strong promoters and a chloramphenicol resistance marker. The SOE PCR method is also described in patent application WO 2003/095658.

The gene was expressed under the control of a triple promoter system (as described in WO 99/43835), consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis cryIIIA promoter including stabilizing sequence.

The gene was expressed with a Bacillus clausii secretion signal (encoding the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA) replacing the native secretion signal. The native secretion signals were predicted using the SignalP 3.0 program (Improved prediction of signal peptides: SignalP 3.0, Bendtsen et al. J. Mol. Biol., 340:783-795, 2004) Furthermore, the expression construct results in the addition of a carboxy-terminal poly histidine tail consisting of 6 consecutive histidine residues.

The SOE-PCR product was transformed into Bacillus subtilis and integrated in the chromosome by homologous recombination into the pectate lyase locus. Subsequently, a recombinant Bacillus subtilis clone containing the integrated expression construct was grown in liquid culture. The culture broth was centrifuged (20000×g, 20 min), and the supernatant was carefully decanted from the precipitate and used for purification of the enzyme or directly in enzyme assays.

The mature expressed protein of the recombinant M4 metalloproteases can be determined by N-terminal amino acid sequencing by Edman degradation and intact molecular weight analysis by mass spectroscopy of the purified protein (Peptide and protein analysis with mass spectrometry, Trauger et al. Spectroscopy, Volume 16 (2002), Issue 1, Pages 15-28.)

EXAMPLE 2 Adding a Protease According to the Invention with Improved Free Amino Nitrogen (FAN) Generation during Small Scale Mashing

The proteases according to the invention (prepared according to Example 1) were compared to the protease Neutrase™ (Novozymes NS) using the following procedure:

-   1. Add 5 g corn starch to 100 mL Blue Cap bottles with magnetic     stirrer. -   2. Grind the malt (from Danish Malting Group (Prod nr 2012-0646)) at     gap 0.2 mm (Bühler mill) and weigh out 5 g in weighing plastic cups. -   3. Add 25 mL 95° C. H₂O, 300 μL CaCl₂ (0.2 M), and 300 ppm Termamyl™     (Novozymes NS) to each bottle with 5 g of corn starch -   4. Do decoction according to mashing profile (see Table 1 below). -   5. Cool down to 50° C. by adding ice or cold water in the water     bath, add 5 g of malt, 25 mL 52° C. H₂O, 0.3 mL CaCl₂, and protease     according to set-up (5, 10, and 15 mg enzyme protein/kg grist) to     each blue cap bottle. -   6. Ready for mashing, set time, and do the mashing manually by     setting the temperature on the water bath. -   7. Cool down to 30° C. and filtrate with small funnels, 50 mL     volumetric cylinders and folded filters (Whatman 597½, ø 185 mm). -   8. Measure the level of Free Amino Nitrogen using NOPA assay and a     Gallery Plus. (The

NOPA assay was Alpha-Amino Nitrogen (NOPA) test kit from Thermo Fisher Scientific (Cat. No. 984342)).

TABLE 1 Mashing profiles: Temperature Time [° C.] [min.] Corn Starch - decoction: 95 30 Malt and Corn mashing without a protein rest: 63 50 72 20 78 15 20 —

Results:

TABLE 2 Δ FAN results without a protein rest: (malt gave a FAN reference level of 105 ppm) 5 mg EP/ 10 mg EP/ 15 mg EP/ Protease: kg grist kg grist kg grist FAN results with Neutrase 8 ppm 12 ppm 18 ppm FAN results with SEQ ID 35 ppm 42 ppm 55 ppm NO: 3 (mature part) FAN results with SEQ ID 16 ppm 24 ppm 29 ppm NO: 10 (mature part) FAN results with SEQ ID 34 ppm 50 ppm 55 ppm NO: 1 (mature part) FAN results with SEQ ID 29 ppm 38 ppm 43 ppm NO: 5 (mature part) FAN results with SEQ ID 28 ppm 44 ppm 49 ppm NO: 2 (mature part) FAN results with SEQ ID 30 ppm 38 ppm 45 ppm NO: 4 (mature part) FAN results with SEQ ID 21 ppm 34 ppm 41 ppm NO: 6 (mature part) FAN results with SEQ ID 15 ppm 26 ppm 32 ppm NO: 9 (mature part) FAN results with SEQ ID 21 ppm 32 ppm 44 ppm NO: 8 (mature part) FAN results with SEQ ID 10 ppm 22 ppm 24 ppm NO: 11 (mature part) FAN results with SEQ ID 18 ppm 32 ppm 46 ppm NO: 7 (mature part)

It can be seen from Table 2 that the M4 metalloproteases according to the invention give surprisingly more FAN than Neutrase. 

1. A method of preparing a wort with an increased level of free amino nitrogen (FAN) comprising: a) preparing a mash from a grist comprising malt and/or adjunct; and b) adding an M4 metalloprotease obtainable from Actinobacteria; and wherein the amount of free amino nitrogen (FAN) in the wort is increased as compared to a wort produced in the absence of the M4 metalloprotease.
 2. The method according to claim 1, wherein the M4 metalloprotease has at least 80% sequence identity to the mature polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.
 3. The method according to claim 1, wherein the M4 metalloprotease is a variant of the polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11 comprising a substitution, deletion, and/or insertion at one or more positions.
 4. The method according to claim 1, wherein the M4 metalloprotease consists of the polypeptide of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.
 5. The method according to claim 1, wherein the M4 metalloprotease is added to the mash or to the wort.
 6. The method according to claim 1, further comprising adding an alpha amylase to the mash.
 7. The method according to claim 1, further comprising adding a beta glucanase to the mash.
 8. The method according to claim 1, further comprising adding a pullulanase to the mash.
 9. The method according to claim 1, further comprising adding a xylanase to the mash.
 10. The method according to claim 1, further comprising adding a lipase to the mash.
 11. The method according to claim 1, wherein the M4 metalloprotease in an amount of 1-100 mg enzyme protein per kg grist.
 12. The method according to claim 1, wherein the grist comprises at least 10% (w/w) adjunct.
 13. The method according to claim 1, wherein the adjunct is selected from the group consisting of barley, rice, corn, wheat, sorghum and cassava.
 14. The method according to claim 1, further comprising fermenting the wort to obtain a beer.
 15. The method according to claim 1, wherein the mashing is done in the absence of a protein rest.
 16. The method according to claim 1, wherein the amount of free amino nitrogen (FAN) is increased by at least 20% when the protease according to the invention in an amount of 10 mg Enzyme Protein per kg grist as compared to a wort produced in the absence of the M4 metalloprotease.
 17. (canceled) 